Understanding the impacts of coastal deoxygenation in nitrogen dynamics: an observational analysis

Biological production and outgassing of greenhouse gasses (GHG) in Eastern Boundary Upwelling Systems (EBUS) are vital for fishing productivity and climate regulation. This study examines temporal variability of biogeochemical and oceanographic variables, focusing on dissolved oxygen (DO), nitrate, nitrogen deficit (N deficit), nitrous oxide (N2O) and air-sea N2O flux. This analysis is based on monthly observations from 2000 to 2023 in a region of intense seasonal coastal upwelling off central Chile (36°S). Strong correlations are estimated among N2O concentrations and N deficit in the 30–80 m layer, and N2O air-sea fluxes with the proportion of hypoxic water (4 < DO < 89 µmol L−1) in the water column, suggesting that N2O accumulation and its exchange are mainly associated with partial denitrification. Furthermore, we observe interannual variability in concentrations and inventories in the water column of DO, nitrate, N deficit, as well as air-sea N2O fluxes in both downwelling and upwelling seasons. These variabilities are not associated with El Niño-Southern Oscillation (ENSO) indices but are related to interannual differences in upwelling intensity. The time series reveals significant nitrate removal and N2O accumulation in both mid and bottom layers, occurring at rates of 1.5 µmol L−1 and 2.9 nmol L−1 per decade, respectively. Particularly significant is the increase over the past two decades of air-sea N2O fluxes at a rate of 2.9 µmol m−2 d−1 per decade. These observations suggest that changes in the EBUS, such as intensification of upwelling and the prevalence of hypoxic waters may have implications for N2O emissions and fixed nitrogen loss, potentially influencing coastal productivity and climate.

biogeochemical cycling of bioelements in Eastern Boundary Upwelling Systems (EBUS) 1 .These impacts include changes in inorganic nutrient concentration and GHG production (CO 2 , N 2 O and CH 4 ) due to DO levels affecting microbial communities involved in carbon (C), nitrogen (N) and phosphorus (P) cycles 2,3 .N cycling involves microbial transformation of organic and inorganic (DIN: NO 3 − , NO 2 − and NH 4 + ) N compounds, including gases such as N 2 and N 2 O. Microorganisms driving these reactions are sensitive to environmental DO levels, and have crucial roles in determining the rates of key processes as nitrification (aerobic NH 4 + and NO 2 − oxidation), denitrification and anammox (anaerobic ammonium oxidation) 4,5 .Denitrification is the dominant pathway for fixed N loss in the EBUS whereas anammox constitutes a minor fixed N loss pathway 3,6 .Fixed N loss results in the depletion of DIN relative to phosphate altering the ratio of nutrient supply to primary producers 4 .
N 2 O, an important GHG, is produced (accumulated) in the EBUS under variable DO levels.N 2 O primarily results from the reduction of NO 3 − and NO 2 − during partial denitrification 7 .Additionally, aerobic NH 4 + oxidation by bacteria and archaea contributes to N 2 O production 8,9 .Conversely, N 2 O consumption, involving its reduction to N 2 , occurs in anoxic environments 4,10 .The EBUS, characterized by strong DO gradients, facilitates these N transformations under both oxic and suboxic conditions, leading to high N 2 O supersaturation and subsequent outgassing towards the atmosphere 9,[11][12][13] .
DO regulates microbial abundance and activity in N cycling pathways at various temporal scales, including the seasonal 14,15 , interannual e.g.El Niño-Southern Oscillation or ENSO 16 , and decadal variability 17 .Moreover, climate change impacts primary productivity and the strength of the biological carbon pump 18 .It is anticipated to enhance wind-driven upwelling in EBUS, modify El Niño-Southern Oscillation (ENSO) events, and exacerbate ocean deoxygenation and hypoxic events due to factors such as ocean warming, stratification, and acidification 1,[19][20][21][22] .
While several biogeochemical models project a substantial reduction in DO levels throughout this century, there is a notable scarcity of long-term observational data confirming coastal deoxygenation trends, as evidenced in the Humboldt Current Systems (HCS) [23][24][25] and California Current Systems (CalCS) 26 .The consequences of this DO reduction, coupled with intensified upwelling on fixed N loss and the accumulation/depletion of N 2 O have been hypothesized but are still inadequately resolved and may vary [27][28][29][30][31] .
Air-sea N 2 O flux exhibits variability due to changes in solubility, wind intensity, the presence of surfactants on the surface, and N 2 O concentrations in both the atmosphere and the surface ocean 32 .In the ocean, this variability is closely tied to microbial N 2 O production, encompassing processes such as nitrification in the mixed layer and denitrification mainly in Oxygen Minimum Zones (OMZs).Considerations for future climatic scenarios should include alterations in microbial community structure due to ocean warming, lower pH impacting the NH 3 -NH 4 + equilibrium and deoxygenation 4,5,32 .
In the HCS, coastal deoxygenation and water mass distribution changes, due to the intensification of upwelling favorable winds, that lead to OMZs being closer to the surface, are processes of particular interest 25 As deoxygenation arises from a complex interplay of oceanographic and biogeochemical processes, the superposition of these mechanisms complicates the attribution to any specific driver.The net N 2 O flux across air-sea interface and denitrification contribution appear to be significantly higher under the impacts of climate change on the ocean 30 .This suggests the existence of positive feedbacks between DO changes induced by climate change and microbial N 2 O emission pathways 33 .These feedbacks are substantial enough to account for the observed acceleration in the N 2 O production rate over the next century 9 .
The main question that inspires this research is: What are the implications of observed temporal (seasonal and interannual) variations in oceanographic/biogeochemical parameters, for N 2 O emissions and fixed N loss, in coastal upwelling regions?.The focus of this research is on understanding the oceanographic conditions and specific biogeochemical mechanisms driving N 2 O accumulation and fixed N loss in the upwelling system off central Chile, particularly in relation to hypoxic water dynamics.

Study area
The study area is located on the 40-km-wide continental shelf off central Chile at 36.51° S, 73.13°W, where a strong seasonal coastal upwelling occurs 34 .There, two water masses are present, a surface water mass of relatively fresh (Salinity < 33.8 PSU), oxygenated (> 200 µmol L −1 l) and nutrient poor (NO 3 − < 4 µmol L −1 ) water of subantarctic origin (SAAW) and equatorward flowing by the HCS, and a subsurface water mass with high salinity (34.9 PSU) oxygen-depleted (4.4-44 µmol L −1 ) and nutrient-rich (20 < NO 3 − < 40 µmol L −1 ) corresponding to equatorial subsurface water (ESSW) and flowing poleward by the Peru Chile undercurrent 35 .When winds favorable to coastal upwelling stress the ocean surface, the SAAW is displaced westward and the ESSW shoals and even reaches the surface 25,36 .
The sampling station (time series station or TSS 18) is located at 92 m depth isobath at 18 nm from the coast (Fig. Supplementary 1) and has been sampled monthly since 2002 and quarterly from 1997 to 2002 as part of time series study of Concepcion University (Chile), constituting one of the few existing long-term monitoring programs with monthly or quarterly sampling cruises 25,[37][38][39] .Our measurements and estimations cover a period of over more than two decades for physical variables (1997-2023) and nutrients, DO and GHGs including N 2 O  (2002-2023).

Sampling and analytical methods
Seawater samples were collected with Niskin bottles from various depths and samples were used to determine nutrient and GHG concentrations.This dataset was already partially analyzed to elucidate DO variability over the past 20 years 25 .Sampling and analytical methods related to N 2 O, and nutrients are described by Farías et al 12 .Calibration procedures, error and uncertainty of N 2 O measurements are presented in Wilson et al 40 .; for nutrient (NO 3 − , NO 2 -and HPO 3 − ) filtered seawater was collected during the whole sampling period and analyzed using manual (1997-2007) and automatic (2008-present) colorimetric methods according to Grasshoff et al. 41 (more information see 12 ); whereas NH 4 + was determined without filtration by fluorometric method 42 .

Data analyses
DIN was estimated as the sum of NO + relative to the other N inorganic forms is very small and has almost no effect on DIN where the main driver is NO 3 − .
N deficit (Eq.2) and N* (Eq. 3) proposed by Broecker & Peng (1982) and Gruber and Sarmiento (1997) 43,44 , respectively, was estimated as: (1) www.nature.com/scientificreports/air-sea N 2 O fluxes were estimated according to: where kw is the transfer velocity from the surface water to the atmosphere, as a function of wind speed, temperature, and salinity from the mixed layer depth (MLD), Cw is the mean N 2 O concentration in the MLD and Ceq is the gas concentration in the MLD expected to be in equilibrium with the atmosphere, according to Weis and Price 45 .Transfer gas velocity Kw as a function of wind speed was based on Wanninkhof (2014) or W2014 46 and compared with that of Wanninkhof (1992) or W92 47 (see Supplementary methods).Climatologies were calculated by the Fast Fourier transform method fitting the seasonal frequency harmonics for each variable obtained in the TSS.Then the climatology was subtracted from the time series to obtain the anomaly.To quantify the proportion of hypoxic waters, present at the sampling location the proportion of the sampled water column under a certain threshold was computed as the total sampling depth minus the shallowest register of DO < threshold.Four DO thresholds were defined to then find the best correlation with N 2 O concentrations, these were 89, 22, 11 and 4.4 µmol L −1 .
Oceanic and coastal episodes of El Niño (EN) and La Niña (LN) were detected using two indexes, the Oceanic Niño Index (ONI) and the coastal El Niño index (ICEN) (see Supplementary methods).Some outliers were removed from physical and nutrient data, particularly from the start of the time series where anomalous values were found.In addition, to assess variability among years, the dataset from 2002 to 2023 was divided into 21 seasons, from September to March (upwelling favorable season) and from April to August (non-upwelling season).Cumulative alongshore (south-north) wind stress for each season was obtained from the cumulative sum of wind stress from the start to finish of each cycle.As in de la Maza and Farias 25 , wind stress was calculated according to Nelson et al. 48for u (cross-shore) and v (alongshore) components using wind speed data from Carriel Sur weather station and ERA5 from the European Center for Medium-Range Weather Forecasts (see Supplementary methods).

Seasonal and interannual variability in nutrient and N 2 O concentrations, N deficit and N 2 O fluxes
The EBUS located in the Pacific Ocean exhibit distinct temporal regimes based on latitude and proximity to the equatorial band, in which they are sited.The EBUS in mid latitudes (30°-45°S) are characterized by a strong seasonality, primarily influenced by the latitudinal migration of high pressure centers (anticyclones) that drive their wind regimes 34,49 .Off central Chile, the South Pacific anticyclone (SPA) drives local winds along the coast and shifts the vertical distribution of the SAAW and ESSW.In the austral fall-winter (April-August), the SPA undergoes a northward migration, giving rise to frequent midlatitude cyclones.This leads to weaker or even northerly winds and a coastal downwelling.Conversely, during spring-summer (September to March), the SPA's southward displacement induces prevailing equatorward alongshore winds, fostering coastal upwelling 50 .This upwelling laterally and vertically transports the ESSW over the continental shelf, where our long TSS is situated (Fig. Supplementary 1), fostering high rates of primary productivity 51 and modulating DO variability and air-sea N 2 O fluxes 12,25 .Table 1 presents basic statistics of all variables and parameter estimates for the 2002-2023 TSS; for most of the variables and estimates, the median and average estimates are very close values, except for N 2 O (2)

N species content and N deficit
Temporal variability of N 2 O concentrations reveal a marked seasonality (Fig. 1a, left panel) with high interannual differences (Fig. 1a, right panel), ranging from 2.34 to 492 nmol L −1 along the time series (mean ± SD = 30.76± 31.33 nmol L −1 ).Low N 2 O levels occur in fall-winter and increase in spring-summer.The highest values consistently occur under hypoxic conditions of 22 < DO < 11 μmol L −1 , the threshold that correlates most strongly with N 2 O in the MLD during the upwelling period (Fig. 1a and e).N 2 O accumulations contrast with low N 2 O levels in bottom water close to the sediments during late summer, occasionally reaching sub-saturated levels around 40% (Fig. 1a).
A similar accumulation/depletion pattern is observed for NO 3 − (Fig. 1b), exhibiting a clear seasonality which is opposite to DO. Lower NO 3 − levels are associated with oxygenated waters, while higher levels are linked to hypoxic/suboxic waters during downwelling and upwelling seasons, respectively.This cycle aligns with the seasonal influence of the ESSW demarcated by the 26.2 isopycnal 25 .The bottom waters during late summer (January and March) exhibit a noteworthy NO 3 − depletion, with levels lower than expected for the ESSW.This suggests a substantial NO 3 − consumption under suboxic conditions, occurring either in the bottom water or in the sediments 52 .This corresponds with the N 2 O depletion (Fig. 1a), indicating that canonical denitrification www.nature.com/scientificreports/(the complete and sequential reduction of NO 3 − to N 2 ) occurs 4 .NO 2 − distribution over time generally remains below 0.2 µmol L −1 throughout the water column, except near the sediments where higher levels (up to 9.73 µmol L −1 ) are observed, particularly in summer (Fig. 1c), coinciding with suboxic conditions (Fig. 1e).NH 4 + shows a similar distribution as NO 2 − (data not shown).The inorganic nutrient N∶P ratios consistently fall below than the expected Redfield ratio 53 in both surface (9.04 ± 1.85) and subsurface (10.15 ± 2.74) layers, whereas N deficit, ranging from 25.60 to − 45.3, increases with depth (Fig. 1d).The maximum N deficits up to − 45.3 µmol L −1 (mean ± SD: -13.20 ± 8.29) are primarily influenced by the lateral and vertical advection of denitrified water (ESSW) poleward transported by the Peru-Chile undercurrent 35 .This is coupled with local dissimilative NO 3 − reduction during anaerobic organic matter mineralization in bottom waters and sediments.Also, N* values show a similar range (mean ± SD: -10.41 ± 8.44 µmol L −1 ), indicating that denitrification predominates over N fixation 44 .As expected, N deficit strongly and negatively correlates with NO 3 − content and the volume of hypoxic waters at various threshold levels (Fig. 2).Notably, the highest correlations between these parameters are identified with DO levels around 11 and 22 µmol L −1 (from 50-80 m depth).

N 2 O exchanges across the air-sea interface
The EBUS represent significant sources of N 2 O to the atmosphere, and their emission rates exhibit spatial and temporal variations influenced by specific atmospheric and oceanographic conditions 54,55 .A comprehensive understanding of this variability is crucial for precise regional and global assessments of N 2 O emissions.It is important to note that the meteorological station at Carrier Sur airport has a continental location (close to the coast), implying a retarding or frictional force on the wind that could impact on wind strength.Results reported by Wong et al. 58 , who conducted an ERA-5 Wind Data validation in the study area, suggest that ERA5 can reproduce the weather conditions at Carriel Sur weather station (r2 = 0.58) with a small bias (0.72 m s −1 ).It is noteworthy that air-sea gas flux estimates scale exponentially with wind speed 46 ), indicating that slightly higher wind speeds offshore may result in significantly higher gas flux rates.By comparing the continental measurements of Carriel sur and the offshore pixel of ERA-5 this dependence is considered providing a range of estimates, Carriel sur being the most conservative of the two.Much temporal variability in air-sea N 2 O fluxes is due to the presence of N 2 O flux hot moments, characterized by disproportionately high N 2 O emission exclusively during the summertime (Fig. 3b).Hot moments are not necessarily associated with the highest upwelling favorable winds (Fig. 3a) but correspond to high surface N 2 O inventories (Fig. 3c) but low for NO 3 − (Fig. 3d); the latter may be an indication of a strong consumption www.nature.com/scientificreports/by phytoplanktonic assimilation.In addition, hot moments, previously described in the TSS 12 , match with N 2 O accumulations in 15-50 m depth and high chlorophyll-a levels.This suggests that an intense microbial activity accompanies the development of such accumulations, and that part of the N 2 O exchanged with the atmosphere may come from the surface.This surface N 2 O is likely produced by nitrifying bacteria coupled with high rates of particulate organic matter accumulation and NH 4 + regeneration 8 .A significant proportion of N 2 O originates from the mid-bottom layer, which is identified as the primary source of N 2 O exchanges with the atmosphere (see below).This layer is characterized by DO concentrations below 22 µmol L −1 .However, it is important to note that the hot moments do not consistently coincide with the periods of maximum hypoxic volumes (Fig. 3e).
The occurrence of these events, totaling 11 in number, introduces significant variability in air-sea N2O flux.Specifically, when excluding these 'hot moments, ' mean N2O fluxes decrease by 33%.We believe that the presence of N 2 O hot moments may be more frequent than currently observed, especially if continuous observation of the surface ocean were maintained.This underscores the importance of synoptic and high-frequency variability 12 and emphasizes the need to consider such factors when estimating regional N 2 O fluxes associated with the EBUS the EBUS.
Strong positive correlations are found among N 2 O fluxes, N 2 O contents at mid-bottom (30-80 m depth) layers, wind stress and the proportion of hypoxia water at various levels (< 89, 22, 11 and 4.4 µmol L −1 ) (Fig. 2).The N 2 O content exhibits the strongest correlation when 4 < DO < 89 μmol L −1 , indicating that the hypoxic range plays a significant role in controlling N 2 O levels in the water column.N 2 O flux also correlates with the increase in the N deficit and the accumulation of NO 2 − (Fig. 2), indicating that N 2 O accumulation and its subsequent exchange primarily proceeds via partial denitrification under hypoxic levels (< 4 µmol L −1 ).The differential sensitivity of N 2 O, NO 3 -and NO 2 − , to hypoxic DO levels is depicted in Fig. Supplementary 2. Recent research, based on experimental data, has revealed that the dominant N 2 O source in oxygen deficient waters is NO 3 − reduction where rates of NO 3 − reduction are found to be one to two orders of magnitude higher than those of NH 4 + oxidation 9,10 .Given the current alterations in intensity and timing of wind within EBUS, which vary widely according to region 59 ; changes in upwelling dynamics may influence the proportion of hypoxic/suboxic waters 25 and therefore the N 2 O balance between production and its consumption.However, N 2 O cycling rates in suboxic regions appear to be an order of magnitude higher than predicted by current models 30 .The rapid rate of N 2 O cycling coupled to an expected expansion of OMZs imply future increases in N 2 O emissions, representing positive feedback of the global marine N 2 O sources.

Variability among annual cycles of biogeochemical variables and drivers
Long time series studies enable us to discern the influence of low frequency climate processes such as the ENSO or the Pacific Decadal Oscillation (PDO) on physical and biogeochemical processes in the EBUS.The ENSO acts as a significant remote forcing influencing the HCS through atmospheric teleconnections and ocean currents, including poleward propagating coastal Kelvin waves 60,61 .ENSO impacts various oceanic parameters such as sea surface temperature (SST), sea level, thermocline depth, surface and subsurface current flows, and upper ocean properties.In terms of biogeochemical processes, the ENSO is expected to induce changes in nutrient availability (N deficit) and primary productivity as thermocline deepens and the wind weakens 62 .But the responses in physical and biogeochemical properties differ markedly between the ENSO events and they even have different responses along the latitudinal gradient as those reported along the CalCS and the HCS 23,[63][64][65][66] .
At low latitudes, EBUS display a significant sensitivity of N 2 O content and air-sea N 2 O flux in response to ENSO 9,13,16 .In literature, surface N 2 O supersaturation in the shelf area during the 2015 El Niño was observed to be nearly an order of magnitude lower than non-El Niño years, implying a significant reduction in air-sea N 2 O efflux (75-95%) 9 .The coupling between physics and biogeochemistry varies between strong and moderate El Niño events 23,67,68 .During strong El Niño events such as the one that occurred in 1997-1998, Kelvin-waveinduced downwelling conditions switched off upwelling, drastically reducing nutrient availability and increasing oxygenation 67 .In contrast, during moderate and weak El Niño events observed in the post-2000 period, equatorial Kelvin wave activity is relatively weaker 69 , maintaining mean upwelling conditions and producing smaller anomalies in nutrient and DO levels 25,70 .
The study period coincides with three notably recorded strong Pacific EN events as 1997-98, 2015-2016 (Godzilla) and 2017 (coastal EN), which rank among the ten strongest EN events recorded in the last century 71 , as well as other moderate and weak EN events in conjunction with LN events (Fig. Supplementary 3 and Table S1).However, non-significant correlations are identified among central (ONI) and eastern Pacific (ICEN) ENSO indexes and biogeochemical variables when all data are considered (Fig. Supplementary 4).Only weak correlations are found with surface T°C, salinity and DO, even though a prolonged oxygenation event occurred in 1997-1998 25 .There is also no relationship between the ENSO indices with the wind stress or with the N 2 O fluxes, suggesting that at mid-latitudes, biogeochemical properties do not exhibit a noticeable response.In the eastern tropical Pacific (ETP) off Peru, significant anomalies in oceanographic and biogeochemical parameters occur in association with the ENSO due to its proximity to the equatorial Pacific.There, ENSO induces changes in ocean circulation patterns, upwelling strength, and thermocline depth, leading to vertical water mass redistributions 72,73 .These dynamics affect the thickness and extent of equatorial subsurface water (ESSW), which is closely linked to the OMZ underlying Tropical Surface Water (TSW) 74,75 .Fluctuations in the extent of the OMZ in this region result from changes in DO supply primarily from lateral (zonal) margins 36,76 .These shifts in the distribution of oxygen-poor waters may have significant implications for the biogeochemical N cycle, particularly in relation to N 2 O emissions 77 .At mid-latitudes off central Chile, a different distribution of water masses is observed respect to tropical regions, with ESSW moving southwards along the Peru-Chile countercurrent, surrounded above and below by well-oxygenated waters such as Subantarctic SAAW and Antarctic Intermediate Water (AIAW).
The dominant meridional transport in mid-latitudes, along with the different distribution of water masses, may explain the lack of correlation between biogeochemical parameters and ENSO.
To disentangle interannual variability, we separate and compare 21 upwelling and downwelling seasons from the beginning of the upwelling season (Sept.) to the end of downwelling (Aug.).To synthesize information for each season and annual cycle, cumulative alongshore wind stress along with nutrient and N 2 O inventories by the surface and mid-bottom layers are shown in Fig. 4 and Table S1.www.nature.com/scientificreports/involves an inverse relationship between mid-bottom N deficit and ONI index with the other variables.This aligns with the expected for upwelling induced variability.PC1 accounts for a substantial proportion of the total variance with 33.7% for downwelling, 41.8% upwelling and 57.8% when both cycles are considered.
Variance distributions for each variable within PC1 are provided in Table 2. PC1 encompasses over 80% and above 70% of air-sea N 2 O flux and surface N 2 O inventory variability, respectively.Differences between upwelling and downwelling seasons emerge in the variance of hypoxic proportion and N deficit within the first component.These N 2 O fluxes are approximately 6-7 times higher during upwelling seasons compared to downwelling seasons (Fig. 4b).Such distinctions between seasons collectively contribute to the observed fluctuations in aggregated seasons, encompassing both inter-seasonal variations and interannual variability.The interannual differences between upwelling seasons are driven by the variability of N 2 O levels in MLD and its air-sea fluxes, hypoxic proportion and mid-bottom N 2 O inventory, with a small incidence of cumulative wind stress and N deficit, while ENSO (ONI) is almost uncorrelated.For downwelling seasons, the differences lie in N 2 O fluxes, N 2 O levels in the MLD and Mid Bottom layers.Counterintuitively, year to year differences in wind stress during downwelling season are more relevant than in the upwelling season, where due to the combined effect of upwelling and enhanced exchange due to wind intensity, a greater ponderation of this variable was expected.
These findings suggest notable interannual variations in air-sea N 2 O fluxes, with distinct controlling factors for each season.During the spring-summer or upwelling season, the highest air-sea N 2 O fluxes are estimated, and variations in the intensity and frequency of upwelling events significantly influence these observed differences.In contrast, during autumn-winter, the heightened presence of the oxygenated SAAW (respect to the ESSW) along with downwelling processes, serves to curtail the N 2 O exchange and production of N 2 O.Other biogeochemical processes should be underlying, particularly the preponderance of reductive processes (denitrification) that dominate in spring and summer, in contrast to oxidative processes (nitrification) that prevail in fall-winter.In fact, during the downwelling season, a greater diversity of activity nitrifying archaea and higher nitrification rates have been recorded in the area affecting N recycling 78,79 .

Interannual trends in nutrients (N deficit), N 2 O content and its air -sea fluxes
Temporal trends of biogeochemical variables and indices estimated for different layers (the MLD: 0-10 mm, subsurface: 11-29 m where oxyline and pycnocline are located, mid 30-65 m, and bottom 66-80 m) are presented in Table 3.The NO 3 − trend indicates that this nutrient is removed in mid and bottom layers at significant rates about 0.15 µmol L −1 y −1 , where DO shows the highest consumption rates (Table 3).This rate corresponds to a NO 3 − loss per decade of 1.5 µmol L −1 , a value that can be significant for the ecosystem if the fixed N loss persists for the next decades 80 .This NO 3 − reduction corresponds to N deficit and N* indices, which increase similarly, indicating that NO 3 − and NO 2 − is being reduced to N 2 O/N 2 .On the other hand, in the same layers, an accumulation of NO 2 − at rates ranging from 0.11 to 0.22 µmol L −1 decade −1 is estimated, only 7% of the NO 3 − reduction.This small NO 2 − accumulation indicates that NO 2 − continue to be reduced or oxidized through processes already described in the study area such as dissimilative NO 2 − reduction, aerobic NO 2 − oxidation (nitrification), anaerobic NH 4 + oxidation (anammox) and even nitrifier denitrification, where NO 2 − is used as electron acceptor or donor 81,82 .Conversely, N 2 O, an intermediate of denitrification or a by-product of nitrification, accumulates in the mid layer at rates of 0.14 nmol L −1 y −1 , but it is consumed in the bottom layer (− 0.07 nmol L −1 y −1 ) (Table 3).These trends patterns are expected based on the DO ranges observed in each layer and the differential sensitivity of the enzymes involved in the sequential reduction of NO 3 − to N 2 5 .These intricate observed patterns underscore the complex interactions between DO availability and N cycling processes, emphasizing the influence of seasonal and interannual variability on the biogeochemical processes in the EBUS.The findings shed light on the mechanisms underlying N 2 O dynamics and highlight the significance of DO levels in governing N transformations in the marine ecosystems.
The reduction of NO 3 − concentration and the increase of N deficit in more than two decades could indeed have significant consequences on primary productivity and the composition of the phytoplankton community, subsequently impacting marine food webs.The decrease in the inorganic N* and N deficit could lead to N limitation in the system, affecting nutrient recycling processes such as the POM remineralization and variations in the N:P ratio of POM 83 ; these changes could further impact nutrient availability, overall productivity and phytoplankton composition, favoring certain phytoplanktonic species better adapted to these altered nutrient proportions 84 , and/or displaying environmental and physiological acclimation responses (e.g., cellular macronutrient contents 80,85  most relevant finding is that in 20 years, the area has increased its N 2 O emission by 1.7%, a much higher rate than that reported for the global ocean 89 .Moreover, if we take into consideration that N 2 O hot moments may occur more frequently than observed during this study's sampling cycle (at intervals of 30 days or more), the N 2 O emission rate could be higher.

Conclusions and perspectives
Strong positive correlations found between N 2 O contents at mid layers along with the proportion of hypoxic water (4 < DO < 89 µmol L −1 ), indicate that this hypoxic range plays a significant role in controlling N 2 O levels mainly via partial denitrification.Given the accelerated deoxygenation observed in the EBUS such as central Chile 25 , the accumulation of N 2 O could be greater in the next decades.
In coastal upwelling areas extending onto the continental shelf, hypoxic conditions often prevail in the midwaters, fostering conditions conducive to incomplete denitrification and subsequent N 2 O production.However, if DO levels decrease sufficiently (< 4 µmol L −1 ) and expand, dissimilative N 2 O consumption can occur through strictly anaerobic microbial processes 83 .Therefore, improved observational data and biogeochemical models are imperative to capture the variability in N 2 O production/consumption rates in the mid and long term 90 .
Despite ENSO being the main driver of interannual variability in the HCS, no significant correlations were found between ICEN and ONI indices with air-sea N 2 O flux.However, there is high variability among years, driven mostly by differences in cumulative wind stress, the presence of upwelled hypoxic waters with high N 2 O concentrations near the surface and the occurrence of N 2 O hot moments.These differences are exacerbated in the upwelling season as the N 2 O air-sea fluxes are up to 7 times higher and hot moments are observed exclusively during this period of the year.
Our findings reveal an increase in air-sea N 2 O flux over the study period, with rates of 0.29 µmol m −2 d −1 y −1 .Furthermore, the N 2 O exchange rate is notably influenced by the existence of hot moments, indicating an accumulation of N 2 O in surface waters.This accumulation is strongly correlated with favorable upwelling wind stress.Therefore, understanding the potential increase in marine N 2 O emissions observed in upwelling systems, which implies positive climate feedback, is pivotal for developing effective strategies to mitigate its influence on the Earth's atmosphere.Predictions regarding coastal upwelling towards the end of this century remain uncertain, given the intricate and competing changes in upwelling intensity, source-water chemistry, and stratification 91 The evolution of marine N 2 O emissions in the twenty-first century in response to anthropogenic climate change remains uncertain [27][28][29] .While some models predict a decline in oceanic N 2 O emissions by 2100 27,28 , this decline seems to be heterogeneous across the global ocean, with N 2 O emissions in the eastern South Pacific predicted to enhance 27 .Uncertainties arise because although more fixed N loss from water column denitrification in expanded OMZs is expected, it is counteracted by less benthic denitrification due to the stratification-induced reduction in organic matter export 27,29 .
Given the ecological and socio-economic significance of EBUS, precise projections of fixed N loss, variability in DIN and in water columns are essential.Consequently, it is crucial to gain a comprehensive understanding of the impacts of hypoxia/suboxia and upwelling intensification on marine ecosystems and coastal communities.To achieve this, further research, long-term monitoring, enhanced data collection, and more sophisticated modeling efforts are indispensable.

Figure 2 .
Figure 2. Heatmap of correlation matrix among biogeochemical variables that include surface and mid-bottom inventories of N 2 O, NO 3 , NO 2 , DO, as well as estimates of N deficit, N:P, N*, N 2 O fluxes, alongshore wind stress and Hypoxic Volumes at 4.4, 11.15, 22.3 and 89.3 (µmol L −1 ) thresholds.Asterisk represents statistical significance.

Figure 4 .
Figure 4. Annual variability among cycles including non-upwelling (april-august) and upwelling (September to March) season of cumulative wind stress (b) air sea N 2 O fluxes (c) monthly averages of surface (d) N 2 O midbottom layer inventories (e) N deficit and Hypoxic proportion (DO < 22.3 µmol l −1 ); 2000-2023 at TSS18.Uw and Dw mean upwelling and downwelling seasons, respectively.

Table 1 .
12scriptive basic statistics for physical and biogeochemical variables throughout the water column.levelanditsair-sea flux.This discrepancy reveals numerous extreme values, referred to as N 2 O hot moments, where N 2 O values exceeded 2 standard deviation units12.Air-sea N 2 O flux without asterisk calculated using Carriel Sur wind speeds; *N 2 O flux with N 2 O hot-moments excluded for calculations using weather station data.Lastly, ** are N 2 O fluxes calculated using ERA5 wind speeds.

Table 2 .
). Percentage of variance contained within the first covariance pattern (PC1) for each original variable.